Centro de Química-Física Molecular and Institute of Nanoscience and Nanotechnology, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
Soft Matter. 2014 Feb 14;10(6):840-50. doi: 10.1039/c3sm52586d.
Understanding the interactions between anionic lipid membranes and amyloidogenic proteins/peptides is key to elucidate the molecular mechanisms underlying the membrane-driven amyloid fiber formation. Here, hen egg-white lysozyme was used as a model protein to test whether this same process also occurs with non-amyloidogenic lipid-binding proteins/peptides. A complementary set of biophysical techniques was employed to study the structure and dynamics of the lipid-lysozyme mixed fibers produced at a low lipid/protein molar ratio that have been proposed earlier to present "amyloid-like" characteristics. The multilamellar architecture of these elongated mesoscopic structures was established by performing time-resolved Förster resonance energy transfer measurements, at both bulk (ensemble) and single-fiber level. The predominantly oligomeric lysozyme and phospholipids were both found to display significantly decreased lateral mobility when embedded in these mixed fibers. Notably, two-photon microscopy of Laurdan revealed that a pronounced membrane surface dehydration/increased molecular interfacial packing was produced exclusively in these elongated mixed supramolecular fibers present in the highly polymorphic samples. Infrared spectroscopic studies of lysozyme in these samples further showed that this protein did not exhibit a rich β-sheet structure characteristic of amyloid fibrils. These results support the conclusion that negatively charged lipid membranes do not have the general ability to trigger amyloid fibril formation of non-amyloidogenic proteins.
理解阴离子脂质膜与淀粉样蛋白/肽之间的相互作用是阐明膜驱动的淀粉样纤维形成的分子机制的关键。在这里，鸡卵清溶菌酶被用作模型蛋白，以测试是否同样的过程也发生在非淀粉样蛋白结合的脂质结合蛋白/肽上。采用了一组互补的生物物理技术来研究在低脂质/蛋白质摩尔比下产生的脂质-溶菌酶混合纤维的结构和动力学，这些混合纤维先前被提出具有“类淀粉样”特征。通过在体相（整体）和单纤维水平上进行时间分辨的Förster 共振能量转移测量，确定了这些伸长的介观结构的多层结构。当嵌入这些混合纤维中时，发现主要是寡聚溶菌酶和磷脂的侧向流动性都显著降低。值得注意的是，Laurdan 的双光子显微镜显示，仅在存在于高度多态性样品中的这些伸长的混合超分子纤维中，才会产生明显的膜表面去水/增加分子界面堆积。这些样品中溶菌酶的红外光谱研究进一步表明，该蛋白没有表现出富含β-折叠结构的特征，这是淀粉样纤维的特征。这些结果支持这样的结论，即带负电荷的脂质膜一般没有触发非淀粉样蛋白形成淀粉样纤维的能力。
